This application is a national phase application of International Patent Appln. No. PCT/GB00/04981 filed Dec. 22, 2000, which designated the United States and was published in English.
The present invention relates to replication incompetent herpes simplex viruses capable of efficiently transferring genes to multiple sites within the nervous system. It also relates to the use of such viruses in the study and treatment of diseases and conditions of the nervous system.
Herpes simplex viruses (HSV) 1 and 2 have often been suggested as a vector for gene delivery to the nervous system and also other cell types (for reviews see Coffin and Latchman 1995, Fink et al. 1996). As a vector HSV has a number of potential advantages in that it naturally enters latency in neurons, providing the possibility of long term gene expression, does not integrate into the host genome, preventing insertional mutagenesis (for example the activation of oncogenes or inactivation of tumour suppressor genes), can accept very large DNA insertions allowing the delivery of multiple genes, is easy to propagate, and can infect a wide variety of other cell types as well as neurons. HSV also has the unique ability among viruses currently under development as vectors in that it can be efficiently transported along nerves to the cell body (usually in the spine) by retrograde axonal transport following an initial peripheral infection. However, while this property of retrograde axonal transport of HSV vectors has been observed with replication competent vectors in the peripheral nervous system (PNS), it has not previously been exploited in vectors used in the central nervous system (CNS), probably due to limitations in the vectors which have previously been available.
While HSVI is highly prevalent in the human population, in the vast majority of cases giving no obvious signs of disease, for use as a vector the virus must be disabled for safety and so as to minimise toxicity to target cells. Various strategies for disablement have been reported including the removal of genes which are unnecessary for growth in vitro but necessary for pathogenesis in vivo. Such genes include those encoding thymidine kinase (TK; Ho and Mokarski 1988), ribonucleotide reductase (Goldstein and Weller 1988) and ICP34.5 (Coffin et al. 1995). However for minimal toxicity it has become apparent that expression of the regulatory immediate early genes ICPO, ICP4, ICP22 and ICP27, which are themselves cytotoxic, must be minimised (Johnson et al. 1994, Johnson et al. 1992, Wu et al. 1996, Samaniego et al. 1998, Krisky et al. 1998). Such reductions in IE gene expression minimise transcription from the vast majority of the 80 or so other genes in the HSV genome. Removal of ICP4 or ICP27 completely prevents virus growth and so such deletions must be complemented in the cells used for virus propagation (e.g. Deluca et al. 1985). Deletion of ICP22 and/or ICPO, while these genes are not absolutely essential for virus growth (Sacks and Shaffer 1987, Stow and Stow 1986, Post and Roizman 1981, Sears et al. 1985), reduces virus titre. Thus for the growth of HSV mutants with multiple IE gene deficiencies, cell lines must be produced which effectively complement deletions from the virus, and for effective growth of viruses with deletions in ICP4, ICP27, ICP22 and ICPO, all these deficiencies would optimally need to be complemented. However as the IE proteins are highly cytotoxic (Johnson et al. 1994), IE gene expression in cell lines must be tightly regulated. This is usually achieved by the use of the homologous IE gene promoters which are relatively inactive in the absence of virus infection (e.g. E5 cells [ICP4], B130/2 cells [ICP27], E26 cells [ICP4+ICP27], F06 cells [ICP4+ICP27+ICPO]; Deluca and Schaffer 1987, Howard et al. 1998, Samaniego et al. 1995, Samaniego et al. 1997). This reduces the problem of IE protein cytotoxicity but still leaves an inherent problem in the generation of cells which are highly effective at complementing multiple IE gene deficiencies.
A second strategy to reduce IE gene expression, rather than deletion of the IE genes themselves, is to include mutations in the gene encoding vmw65. vmw65 is a virion protein which transactivates IE promoters after virus infection (Batterson and Roizman 1983, Pellet et al. 1985), and while an essential structural protein, specific mutations abolish the trans-activating capability of the protein without affecting the structural integrity of the virus (Ace et al. 1989, Smiley and Duncan 1997). These mutations vastly reduce IE gene expression although at high multiplicity or with the inclusion of hexamethylene bisacetamide (HMBA) in the media still allow efficient virus growth in culture (McFarlane et al. 1992).
Thus, for the construction of vector viruses the approach could also be taken of combining mutations in vmw65, which should reduce expression of all the IE genes, with deletion of ICP27 and/or ICP4, the two essential IE genes, giving viruses as above in which overall IE gene expression is minimised. This is the approach we have taken to generate non-toxic HSV vectors in which IE gene expression has been minimised but which can still be grown in culture using cell lines containing the genes encoding ICP27, ICP4 and the equine herpes virus (EHV) homologue of vmw65 (encoded by EHV gene 12; Thomas et al. 1999).
The development of HSV vectors has also required a second problem to be overcome before they can be used to take advantage of the natural lifecycle of the virus in which a latent state is maintained in neurons. This problem results from the finding that in most cases promoters driving genes inserted into an HSV vector genome are rapidly transcriptionally inactivated as the virus enters latency, including the promoter which usually drives the expression of the only HSV transcripts present during latency, the latency associated transcripts (LATs). To solve this problem, a number of approaches have been taken:
First, it was found that a Moloney murine leukaemia virus (MMLV) promoter linked to a fragment of the LAT promoter (xe2x80x98LAP 1xe2x80x99; Goins et al. 1994) and inserted into the gene encoding glycoprotein C (gC) was able to drive expression during latency, although neither LAPI or the MMLV promoter alone, or a number of other promoters linked to LAPI or alone allowed this to occur (Lokensgard et al. 1994). MMLV alone inserted in ICP4 (Dobson et al. 1990, Bloom et al. 1995) or in LAPI (Carpenter and Stevens 1996) however was active, which may be speculated to be due to the proximity of these regions to the endogenous LATs rather than when distant to the LAT region (in gC) as before. In other approaches it was found that LAP2 alone could give expression during latency when inserted in gC (Goins et al. 1994), but this expression was very weak, and that LAP2 linked to LAPI, like MMLV linked to LAPI, could also maintain latent gene expression when inserted in gC (Lokensgard 1997). Finally it was found that insertion of an internal ribosome entry site (IRES) into the 2 Kb LAT allowed expression of a downstream marker gene (with poly A site) during latency (Lachmann and Efstathiou 1997). However while the above approaches have demonstrated latent gene expression in the PNS, as yet latent gene expression in the brain has only been demonstrated in a very small number of transduced cells (Dobson et al. 1990, Bloom et al. 1995, Carpenter and Stevens 1996) and effective replication defective disabled HSV vectors for the long term gene transfer to the brain have not previously been available.
We have taken an alternative approach to the development of promoters allowing latent gene expression from within the context of an HSV vector where we have found that elements of the LAT region can be used to confer a long term activity onto promoters which are not usually active during latency by placing them downstream of a region designated LAT P2 (promoters inserted after HSVI nt 120,219; Genbank file HEICG: W098/30707). For example a cytomegalovirus (CMV) IE promoter and some other promoters including a minimal neuron specific enolase (NSE) promoter and the Moloney murine leukaemia virus (MMLV) LTR promoter placed in such a context remain active during latency, even though when placed elsewhere latent gene expression with such promoters is generally very inefficient.
As discussed above, HSV has the unique property among viruses currently under development as vectors in that it can be efficiently transported along nerves before entering latency in neuronal cell bodies. However it has not previously been found that this property can be exploited for gene delivery in the CNS using disabled herpes vectors, as replication incompetent, herpes vectors so far used in the CNS have not demonstrated such properties as gene delivery has always been limited to the site of inoculation. The present inventors have identified disabled, replication incompetent herpes vectors which allow gene delivery to multiple regions within the brain following vector inoculation to only a single brain region. This results from highly efficient retrograde transport of the vectors to cell bodies at connected sites. The inventors have also identified the properties of the vectors which allows this to occur. Replication incompetent herpes vectors allowing such gene delivery to occur combine the use of highly disabled replication incompetent vector backbones with promoter systems which are capable of directing gene expression in the long term. The surprising finding was made that vectors which were either less disabled than those provided by the invention (ie which potentially showed greater toxicity), or which contain promoter cassettes which are incapable of directing latent gene expression, do not allow such widespread gene delivery to occur, even in the short term. Replication incompetent herpes virus vectors as identified by the present inventors may be used in methods of studying or treating disorders of the central nervous system. In particular such vectors may be used in methods of target validation, for example, by screening genes to identify genes important in a central nervous system disorder.
Accordingly, the present invention provides:
a use of a replication incompetent herpes virus comprising:
(a) a mutation which prevents or reduces the expression of at least two immediate early genes; and
(b) a heterologous gene operably linked to a promoter active during herpes virus latency
in the manufacture of a medicament for use in treating or preventing a central nervous system disorder;
a method of determining whether a gene has an effect on a phenotype associated with a central nervous system disorder or on a cell of the central nervous system which is relevant to a central nervous system disorder, which method comprises:
(i) inoculating into a cell of the central nervous system in vivo or in vitro with a replication incompetent herpes virus comprising:
(a) a mutation which prevents or reduces the expression of at least two immediate early genes; and
(b) a heterologous gene operably linked to a promoter active during herpes virus latency; and
(ii) monitoring a phenotype of said disorder or an effect of expression of said gene on said cell to determine thereby whether said gene has an effect on said cell or said phenotype;
a method of screening genes implicated in a central nervous system disorder to identify a target for gene therapy or for small molecule modulators, which method comprises a method of determining whether a gene influences a phenotype associated with a central nervous system disorder according to the invention;
a method of treating a subject suffering from a disorder of the central nervous system, which method comprises administering a therapeutically effective amount of a replication incompetent herpes virus comprising:
(a) a mutation which prevents or reduces the expression of at least two immediate early genes; and
(b) a heterologous gene operably linked to a promoter active during herpes virus latency
to a subject suffering from a disorder of central nervous system;
The present invention is exemplified using vectors in which immediate early gene expression has been minimised by deletion of ICP27 and ICP4 and with a mutation minimising the transactivating capabilities of vmw65, although other similarly non-toxic vector backbones may be used. The invention is also exemplified using vectors in which the LAT region has been used to confer long term activity onto a promoter not usually active in the long term, although other promoter systems active during latency might be used, including those discussed above (Lokensgard et al. 1994, 1997, Goins et al. 1994, Lachmann and Efstathiou 1997).
The term heterologous gene is intended to embrace any gene not found in the viral genome. The heterologous gene may be any allelic variant of a wild-type gene, or it may be a mutant gene. Heterologous genes are preferably operably linked to a control sequence permitting expression of said heterologous gene in a cell of the nervous system. Viruses of the invention may thus be used to deliver a heterologous gene/genes to cells of the nervous system where it will be expressed in multiple regions following inoculation to anatomically connected site(s).